The purpose of this project is to stuey the organization and regulation of the Punch (Pu) locus in D. melanogaster. The locus encodes the enzyme, GTP cyclohydrolase, the enzyme catalyzing the first reaction in pteridine biosynthesis. Pterins serve as pigments in Drosophila, but also are universally used as enzyme cofactors for reactions that include the synthesis of serotonin and catecholamines. Pterin deficiencies in humans are lethal. Pu is complex in its organization and development expression, as indicated by the generation fo developmentally-specific mutations and by complex patterns of interallelic complementation. Our research suggests that Pu encodes at least three polypeptides, each of which is expressed at a discrete developmental time. Their individual times of expression correspond to periods of know Pu function as assessed by enzyme assays or mutant phenotypes. Complementing lethal and developmentally specific Pu mutations have discrete and predictabfle protein alterations taht correspond to specific interallelic complementation groups. Each lethal complementation groups also corresponds to predictable, and essentially mutually exclusive embryonic lethal phenotypes, with alleles in one group affecting late embryonic lethal phenotypes, with alleles in one group affecting late embryonic functions and alleles of another group affecting very early, patterning functions. Thus, we have discovered direct correlations between genetic domains of the locus, protein products and mutant phenotypes. The proposed experiments are intended to shed light on the molecular basis for the complexity of this locus, and to determine the mechanisms by which Pu is regulated. About 200 kb of the Pu region DNA have been cloned, and a study of the physical organization of the region has begun. The experiments described in this proposal are a continuation of the molecular genetic analysis now in progress. Molecular mapping of rearrangements, deletions and insertions associated with Pu mutations and with mutations in neighboring complementation groups will be completed. Transcripts will be mapped by Northern blot, S1 endonuclease degestion, and primer extension experiments. Libraries of cDNAs are being screened, and isolated cDNA clones will be characterized. Hybrid selection- in vitro translation experiemnts and means of obtaining more specific antibodies to the Pu products are planned. Transcripts in Pu mutants will examined, and in situ hybridization of single- stranded probes to tissue sections is proposed in order to assess Pu expression in developmental times and in tissues where other forms of analysis are not practical.